Accurate isoform discovery with IsoQuant using long reads

Prjibelski, Andrey D.; Mikheenko, Alla; Joglekar, Anoushka; Smetanin, Alexander; Jarroux, Julien; Lapidus, Alla; Tilgner, Hagen U

doi:10.1038/s41587-022-01565-y

Cited by 49 publications

(51 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The poly(A) tail lengths of the reads were estimated using Nanopolish-polyA (v10.2) (https://nanopolish.readthedocs.io/en/latest/quickstart_polya.html, [53]) on the reads previously aligned to the GRCh38.p13 human genome and PR8 reference genome using Minimap2 (v2.12) (https://github.com/lh3/minimap2; [73]) in splice mode. We used the mapping tool Isoquant (v3.3) (https://www.gencodegenes.org/human/, [74]) to assign a human gene to each human read using the GRCh38.p13 human genome and the human gene database gencode.v42. A version with and a version without mitochondrial sequences of both the genes and the genome references were used to delete reads for mitochondrial RNA.…”

Section: Methodsmentioning

confidence: 99%

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Bougon,

Kadijk,

Gallot-Lavallee

et al. 2023

Preprint

View full text Add to dashboard Cite

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their own protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine if NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X effects on host gene expression are not simply additive and that these factors functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including influenza A virus continue to cause annual epidemics with high morbidity and mortality due to limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff – a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the non-structural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterised some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs development of improved live attenuated vaccines.

show abstract

Section: Methodsmentioning

confidence: 99%

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Bougon,

Kadijk,

Gallot-Lavallee

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Oxford Nanopore Technology (ONT) and PacBio HiFi sequencing yielded 250x10 6 and 38x10 6 barcoded long reads respectively for 395 cell clusters (e.g., P56:Thalamus:Replicate1:OPCs) obtained from the short-read analysis pipeline (Methods, Table S2-3). Using recent transcriptdiscovery software 48,49 high accuracy single-cell PacBio reads identified novel splice sites, enhancing the GENCODE annotation by 22.1% (40,184 transcripts). Over 67.3% of mapped, barcoded ONT reads (SD=4.51%, Table S2) represented multi-exonic transcripts with trustworthy splice sites, and ~70% of these ONT transcript models corresponded to annotated or PacBio-derived transcripts (Methods, Fig S1).…”

Section: Resultsmentioning

confidence: 99%

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

Joglekar

Zhang³

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

show abstract

“…In order to ensure the general applicability of scywalker outside of human (or even animal) data, we also generated two plant single-cell data sets (Arabidopsis thaliana), which were analyzed successfully by scywalker, also showing very high correlation with their respective short-read results. Isoform discovery in scywalker is based on IsoQuant, a proven tool showing good results in bulk long-read RNA-seq analysis 14 . In scywalker, the initial discovery is performed without taking cell barcodes into account, i.e., on bulk RNA.…”

Section: Discussionmentioning

confidence: 99%

“…The module ends with creating barcoded FASTQ files where each read has its corrected droplet barcode and UMI sequence added. In the second module, the droplet barcoded reads are aligned to the reference, and isoforms (and genes) are first detected and quantified in a bulk analysis using an adapted version of IsoQuant 14 for the non-organelle chromosomes. Gene counting for organelles is done separately using a specific method because organelles, given their different transcription structure and extreme read counts, often pose problems to isoform callers.…”

Section: Introductionmentioning

confidence: 99%

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

De Rijk,

Watzeels,

Küçükali

et al. 2024

Preprint

View full text Add to dashboard Cite

We introducescywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.

show abstract

Accurate isoform discovery with IsoQuant using long reads

Cited by 49 publications

References 38 publications

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

Contact Info

Product

Resources

About